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HIV drug resistance is a multifactorial phenomenon and constitutes a major concern as it results in therapy failure. The aim of this study was to assess the impact of an amino acid insertion identified at position 33 of the protease gene, derived from samples from three patients under lopinavir therapy, on viral fitness and protease inhibitor (PI) resistance. Successive samples were available from one of the patients for genotypic and phenotypic testing in order to investigate the role of this insertion. The patient had been pretreated with various antiretroviral drugs and showed poor virological response from the point of the acquisition of the mutation onward. The insertion was acquired in the context of a number of other PI mutations and was stable following acquisition. Phenotypic testing revealed reduced susceptibility to various PIs and a reduction of the replicative capacity (RC) of the virus. In the presence of the insertion alone, a decrease of the RC was observed, which seemed to be compensated by the presence of other mutations. The L33ins might have a potential role in PI resistance pathways but further investigation in a larger number of clinical samples is required in order to elucidate this resistance mechanism.

It has been difficult to explain why all HTLV-1 sequences in Salvador, a city in the northeast of Brazil, belong to the Transcontinental (A) subgroup of the Cosmopolitan (a) subtype, since according to historical data the vast majority of slaves brought to Brazil (through Salvador) came from west Africa, where only the western African subgroup (C) has been found. To shed more light on this subject we conducted a phylogenetic analysis of 23 isolates from blood donors of Salvador. DNA was extracted and submitted to a nested PCR for amplification of the entire LTR region. The PCR products were purified and sequenced on an automated sequencer. Neighbor-joining and maximum likelihood phylogenetic analyses were performed. None of the new sequences from Salvador clustered within the West-African subgroup C. Confirming previous results, all sequences belonged to the Transcontinental subgroup (A) of the Cosmopolitan subtype, and clustered in two Latin American clusters. In addition we showed sequences from southern Africa clustering in both Latin American clusters. One of the new sequences is ancestral to the larger Latin American cluster beta due to a duplication of a 12-bp long fragment, a finding that has not been previously described. These findings support the hypothesis that HTLV-1 isolates circulating in Latin America have a closer relationship to South African compared to West-African HTLV-1 strains. The 12-bp-long duplications in one of the sequences has no obvious clinical or biological implications yet.

In Brazil, HTLV-2 has been detected in blood donors, in intravenous drug users (IDUs) from urban areas, and in Amerindians living in the Amazon basin. Of the three main HTLV-2 subtypes (2a, 2b, and 2d) only subtype 2a has been detected in Brazil. However, a molecular variant of subtype 2a (also called HTLV-2c) characterized by an extended Tax protein has been isolated from Brazilian blood donors, IDUs, and Indians. Here, we analyzed HTLV-2 isolates from 10 IDUs and a Chilean woman living in Salvador, Bahia, Brazil. Sequencing of env, pX, and long terminal repeat (LTR) genes demonstrated that 10 of the isolates are related to the Brazilian subtype 2a molecular variant described previously. We show that most HTLV-2a Brazilian strains comprise a phylogenetic group harboring a considerable degree of diversity within the env region but not within the LTR region. Interestingly, we demonstrated for the first time in Brazil the presence of a subtype 2a in IDUs that is closely related to the prototype Mo but distinct from the Brazilian 2a molecular variant. ispartof: AIDS Research and Human Retroviruses vol:19 issue:6 pages:519-23 ispartof: location:United States status: published

213 IN THE DISCUSSION ON THE ORIGIN of the human immunodeficiency virus type 1 (HIV-1) it has been hypothesized by E. Hooper in his book The River that the oral polio vaccine (OPV) could be held responsible for the introduction of the simian immunodeficiency chimpanzee virus (SIVcpz) into humans, leading to its human counterpart, HIV-1.1 This theory suggests that oral polio vaccine stocks would have been prepared in chimpanzee kidney tissue contaminated with SIV. During the African vaccination trials in the 1950s these allegedly SIV-contaminated OPVs would have then been administered to people in what is now the Democratric Republic Congo. However, this OPV/HIV hypothesis has recently lost a great deal of its credibility due to the work of Korber et al.,2 Salemi et al.,3 Rambaut et al.,4 Blancou et al.,5 and Berry et al.6 The first three studies show that HIV-1 group M infection in humans originated before the 1930s and thus predates the oral polio vaccination trials, while the last two studies confirm that macaque kidney tissue had been used for the preparation of the early batches of the OPV, and the absence of chimpanzee mitochondrial (mt)DNA and HIV/SIVCPZ-like sequences in these vaccines. However, as R. Weiss7 mentioned, it could still be argued whether “chimpanzee kidney cells” and bonobo kidney tissue “could have been used locally in Africa to amplify the batches of the OPV,” as insinuated by E. Hooper.1,8,9 As E. Hooper stated in his book, bonobos (Pan paniscus) have been reported earlier by us and others,10,11 to be at the origin of another zoonotic human retrovirus, human T-cell lymphotropic virus type II (HTLV-II). Thus, bonobos and the use of their tissues may pose a potential risk for cross-species transmission of other etiologic agents. SIV infection has been reported in various African monkey species, but no data about the serostatus in bonobos have been published yet. The fact that SIV infection has been described in two subspecies of the bonobo’s closest related simian species, the common chimpanzee or Pan troglodytes,12–14 raises the question of whether bonobos are also a natural host for SIV. This is especially interesting since the natural habitat of the affected P.t. troglodytes and P.t. schweinfurthii subspecies neighbors that of the bonobo, which is situated in the Central Congo basin, between the Congo River, the Lomami and the Kasai/Sankuru Rivers, and the Lake Tumba/Lac Ndombe area (Fig. 1). To assess the prevalence of SIV in bonobos, we have performed a serologic survey of 26 bonobos, of which 14 were wild caught and 12 were born in captivity. Thirteen animals were housed at the Yerkes primate center in Atlanta, Georgia, 7 in a breeding center of the Antwerp Zoo in Belgium, 2 in the Leipzig Zoo in Germany, and 4, saved from poachers, were housed in an orphanage in Congo Brazzaville. The bonobo plasma/serum samples were screened with either the Genetic Systems HIV-1/-2 peptide EIA (Redmond, WA), the Abbott AxSYM HIV-1/2 microparticle EIA (Abbott Laboratories, Delkenheim, Germany), or an in-house SIV antigen ELISA using SIVmac251 purified viral lysate (Advanced Biotechnologies Incorporated, Columbia, MD). All test results were negative. Additional screening of all 26 bonobo samples with the INNOLIA HIV (Innogenetics, Ghent, Belgium) confirmation test, a line immunoblot assay more sensitive for divergent SIV strains than commercial Western blots,15 found no reactivity in all 26 bonobos. Previously, these animals were screened for infection with another simian retrovirus, the simian T-cell lymphotropic virus type I/II (STLV) using an HTLV-I/-II particle agglutination test (Serodia-HTLV, Fujirebio, Japan) and/or an

HIV-1 samples from six patients undergoing diverse anti-HIV therapies possessed the E138A mutation in their reverse transcriptase (RT) genome. Patients were receiving the following therapies: TIBO monotherapy (one patient); zidovudine plus didanosine combination therapy (one); zidovudine monotherapy (one); sequential therapy with zidovudine, then stavudine and finally zalcitabine plus didanosine (one); and two were drug naive. E138K, not E138A, is a known TSAO-specific resistance mutation, emerging under selective pressure in vitro. Our phenotypic data on the patient isolates, confirmed by data on an E138A mutant acquired through in vitro mutagenesis, indicated that an alanine substitution for glutamate at codon 138 of the HIV-1 RT renders the virus TSAO resistant, confirming the importance of this amino acid residue in the activity of TSAO derivatives. In addition, we have demonstrated through phenotypic analysis of the E138A and A98S mutants (after in vitro mutagenesis) that the mutation A98S, found in one of these patients, could be partially responsible for the phenotypic reversal of TSAO resistance. This reversal could be explained by the restoration of a hydrogen bond between 98S and the main-chain residue L349, which compensates for the loss of the E138-G99 main-chain hydrogen bond. As TSAO derivatives have not been used in the clinical setting, the presence of the E138A mutation at a frequency of 6.7% in our study of 90 TSAO-inexperienced HIV-seropositive individuals implies that 138A of the RT must be a natural variant and that the mutant virus is replication competent. Our observations suggest that the E138A mutation may likely arise in patients under the selective pressure of TSAO or related compounds that show a decreased antiviral potency toward the E138A variant.

We have tested the effect of oncostatin M (OSM) on the Tat-mediated trans-activation in a HeLa cell line (HLtat) expressing Tat, using a transfection assay with the LacZ gene under the control of the HIV-1 LTR. Oncostatin M reduced the LacZ expression by 50% at a concentration of 9.5 ng/ml (IC50), which was far below the 50% cytotoxic concentration (CC50 > 400 ng/ml). Although HLtat cells may represent an interesting model for the study of the signal transduction pathway of OSM, this cytokine did not inhibit the tumor necrosis factor (TNF)-dependent activation of the HIV LTR in Molt pNAZ cells or the Tat-mediated trans-activation in HeLa, HeLa-CD4, Hep-II, COS-7, or Jurkat-tat cells. Likewise, OSM did not show any anti-HIV-1 activity in the MT4 cell/MTT assay. Our findings with OSM indicate that, for the screening of HIV Tat inhibitors, care must be taken in selecting a system that not only emulates HIV Tat trans-activation, but is also representative for in vivo-infected cells.

A type-specific serological survey among 1042 random nonneurological outpatients in two cities in the state of Ceara (northeastern Brazil) shows a low prevalence of HTLV-I (0.34% in Fortaleza; 0.44% in Crato) and of HTLV-II (0.34% in Fortaleza; 0% in Crato). Among 62 chronic myelopathic patients seen in Fortaleza 27 patients were found with clinical features of tropical spastic paraparesis (TSP); 10 of 27 were found HTLV-I seropositive (37%; 95% confidence limits, 19-58%). Proviral genome detection by polymerase chain reaction in 5 seropositive and 12 seronegative patients confirmed the serological findings. This excludes HTLV-I or -II infection as a cause in the seronegative TSP patients. The HTLV-positive and -negative patients did not differ clinically and by history, except that seropositives had a longer mean disease duration, a female predominance, and a higher proportion of white Caucasians. In this population with low HTLV-I and HTLV-II prevalences, HTLV-negative TSP is at least as frequent as the HTLV-I-associated TSP.

Sequence analysis can be used to evaluate transmission networks. We have used retrospective samples to examine two HIV-1 transmission networks established by contact tracing. Regions of the HIV-1 region representing segments of gag and env were amplified by RT-PCR from frozen plasma samples and the sequence of each PCR product was determined. Within one of the networks (composed of 38 subjects) we found only a subset of the tested sequence clusters was consistent with the reported epidemiological linkage. Of 15 presumed transmission events where sequence data were available, 9 could be rejected either by subtype mismatch or by phylogenetic tests. In the other network (composed of 89 subjects) we were able to assess sequences for 26 presumed transmission events, 18 of which were rejected based on subtype discordance. Long lags in time between the time of transmission and the time of sequence sampling (ranging from 2 to 18 years) may limit the sensitivity for the detection of sequence linkage. Also, superinfection and incomplete epidemiological information are other factors that will limit the concordance of phylogenetic reconstruction and reported epidemiological linkage.

Epidemiologically-linked HIV-1 transmission cohorts serve as excellent models to study HIV disease progression. The actual relationship between viral variability and HIV disease outcome can be extrapolated only through such rare epidemiologically linked HIV-1-infected cohorts. We present here a cohort of three patients with the source termed donor A (a nonprogressor) and two recipients B and C. Both recipients acquired HIV through blood transfusion from donor A and have progressed to AIDS. By analyzing 15 near full-length HIV- 1 genomes (8.7 kb each genome) from longitudinally collected peripheral blood cell samples (four time points for patient A, four for patient B, and seven from patient C), we were able to demonstrate transmission of HIV from donor A and epidemiologic linkage among members A, B, and C after 10 years of HIV infection. These analyses are novel in demonstrating that HIV-1-infected nonprogressing individuals bear the potential to transmit HIV-1 variants and that HIV variants, which led to a benign disease in a nonprogressor donor, were able to cause disease in other individuals. Overall, these studies highlight the utility of full genome sequencing in establishing epidemiologic linkage in a chronically infected HIV cohort after 10 years of initial infection.

Adherence to highly active antiretroviral therapy (HAART) is crucial, but which aspects of drug-taking behavior are important remain largely unknown. In a prospective observational study, 43 HIV-1-infected patients taking HAART underwent electronic event monitoring (EEM). Taking adherence was defined as the percentage of doses taken compared with the number prescribed, dosing adherence was defined as the percentage of days on which all doses were taken, and timing adherence was defined as the percentage of doses taken within 1 hr of the time prescribed. Drug holidays were defined as periods of no drug intake for24 hr. Cluster analysis, including the four EEM parameters, was used and refined to construct an algorithm to discriminate patients. Patients were categorized as nonadherent if they had a taking adherence of90%, or a dosing adherence of75% and at least 1 drug holiday, or a timing adherence of80% and at least 1 drug holiday, or6 drug holidays per 100 days. All four EEM parameters differed significantly (p0.0001) between the two groups. Adherent patients had a better outcome, as shown by a larger drop in viral load (p = 0.011) and rise in CD4+ cell count (p = 0.035), showing that the algorithm-based categorization is clinically relevant.

We evaluated the predictive value of baseline HIV-1 genotypic resistance mutations for failure of a nucleoside reverse transcriptase inhibitor (NRTI) containing therapy. The change in therapy of 88 HIV-1-infected patients was analyzed retrospectively, relating the genotypic resistance profile at baseline to the evolution of viral load and CD4+ T cell counts. Genotypic resistance at baseline and at 6 months was evaluated with the LiPA HIV-1 RT, which detects mutations at codons 41, 69, 70, 74, 184, and 215. At 1 to 3 months after change in therapy, patients without preexisting resistance mutations to the new drug (group S) had a significantly better evolution in viral load (reduction of 0.37 log(10)) compared with patients with known preexisting resistance mutation(s) (group R) (increase of 0.08 log(10)). This difference was particularly striking for patients with the baseline M184V mutation and whose treatment was modified by the addition of lamivudine. After 6 months the median difference in viral load evolution between the two groups increased to 0.61 log(10): the viral load of patients of group S was still 0.18 log(10) below baseline while patients of group R had an increase of 0.43 log(10) in viral load above baseline. Changes in CD4+ T cell counts were not significantly different. The evolution in viral load in HIV-1-infected patients with and without baseline resistance mutation(s) toward a newly added NRTI is significantly different at 1-3 months and at 6 months after changing or adding one NRTI.

585 WITHIN THE MAJOR a Mo group of HIV-1, phylogenetic analysis permits subclassification into the subtypes A to J. In 1996, a reference set of HIV-1 sequences representing the different subtypes was proposed and this list has been updated and modified as more full-length HIV-1 genomic sequences, including an increasing number of complex recombinant isolates, have become available.2 An analysis of four full-length HIV-1 genomes from Africa (DJ263, DJ264, HH8793, and SE6165) has proposed a new classification of strains previously defined as subtype G or as containing regions of subtype G.3 Isolates with a characteristic genomic pattern of recombination between subtypes A and G cluster together into a distinct clade exemplified by the strain IbNg. This recombinant virus shows an extensive geographic spread, having been isolated in Nigeria, Djibouti, and, by extrapolation of more limited sequence data from isolates that cluster closely with these examples, in Ivory Coast, the Democratic Republic of Congo (DRC), and Gabon.3,4 It is proposed that such circulating recombinant HIV-1 strains should be designated as a circulating recombinant formso (CRFs) of the virus, rather than as separate subtypes. Thus, sequences of the IbNg CRF type are termed a AG-IBNG.o By contrast, the two novel isolates HH8793 and SE6165, from Kenya and Congo, respectively, carry genomes that appear to be nonrecombinant and cluster together in subtype G. This sequence note presents the full-length genomic sequence of a subtype G HIV-1 originating in Kinshasa, DRC (termed DRCBL). The clinical history of the infected patient and initial subtyping of the virus in gag have been previously reported as isolate BL.5 It has become apparent that classification of HIV-1 on the basis of the analysis of small genomic regions is sometimes inadequateÐparticularly when dealing with isolates of African origin. Complex recombinant forms of HIV1 are increasingly being identified and accurate classification requires the examination of large, continuous regions of the genome. For this reason the entire genomic sequence of the virus was determined from patient peripheral blood mononuclear cells (PBMCs) by a previously described method.6 All reading frames were open and intact, apart from a 7-nucleotide (nt) deletion in pol at position 2948, resulting in a premature termination of the predicted protein product. This deletion was most probably the result of deriving the genomic sequence from a single integrated provirusÐmany of which contain defects. Its presence had no effect on the subsequent phylogenetic analysis. Initial phylogenetic analysis of p17gag, pol, the C4±V4 region of env and nef of DRCBL, by comparison with reference strain sequences, showed clustering in subtype G with a high degree of confidence in all regions (data not shown). Neighborjoining phylogenetic analysis was then performed using fulllength genomic sequences from reference strains of subtypes A to J. DRCBL clustered with subtype G isolates with 100% bootstrap value (Fig. 1). The LTR of DRCBL shows the described structural features of subtype G. Interestingly, the 3-nucleotide TAR bulge has the sequence UUUÐ in common with the isolates SE6165, HH8793, DJ263, DJ264, 92NG083.2,7 92NG003.1,7 and 92RU1318; but not IbNg, which has UCU. To examine DRCBL for evidence of intersubtype recombination, maximum parsimony bootscanning was performed as described with a sliding window of 300 nt advancing in 20-nt increments (Fig. 2). DRCBL was compared with the two reference subtype G strains SE6165 and HH8793 (mentioned above). One reference strain of each subtype was included, excepting E, because the A-type portions of this recombinant sub

In most parts of Europe only a limited number of sporadic cases of HTLV-I infections have been identified. So far, the few cases found in Germany have always been linked to individuals with relations to endemic areas. Here we report the first HTLV-I infection from a German ATL patient without any known risk for HTLV-I infection and with no relations to known endemic areas. The DNA sequence of the provirus was determined, and a phylogenetic analysis based on the LTR sequence established a close relationship with HTLV-I sequences previously found in two Romanian patients. Our data suggest the existence of a previously unrecognized cluster of HTLV-I infections in southeastern or central Europe.

The level of HIV-1 RNA in plasma has become one of the most important markers in the follow-up of HIV-infected patients. Three techniques are commercially available: both the Amplicor HIV Monitor and the NASBA HIV-1 RNA QT are target amplification methods, whereas the Quantiplex HIV RNA assay is a branched DNA signal amplification technique. Detection in both target amplification techniques is based on a single primer pair and a single probe in the gag region, whereas multiple probes capture the pol region of the viral RNA in the branched DNA assay. We investigated the discrepant observation of an undetectable viral load in an immunodeficient pregnant HIV-1-infected patient of African origin with no prior antiretroviral treatment. Although clinical progression was present in this patient with tuberculosis and a low CD4 cell count, viral load determinations with both the Amplicor Monitor and NASBA assays revealed no detectable RNA levels. The presence of HIV-1 RNA in the plasma of the patient was demonstrated by an in-house RNA-PCR. Subsequent HIV-1 RNA quantification with the branched DNA method revealed a high viremia (460,000 copies/ml). DNA sequence analysis of the gag gene identified a subtype G HIV-1 strain (HIV-1BL). To our knowledge this is the first report of a patient harboring an HIV-1 genotype of the main group with a high viral load as quantified by the branched DNA assay, but undetectable with the two commercial HIV RNA amplification techniques because of genetic divergence. In the case of discrepant low viral loads determined by one amplification technique in patients with advanced clinical stage one should use an alternative quantification technique for confirmation.